Surface properties of bacterial cells enable them to be used in the removal of heavy metals. Moreover, the inactivation of bacterial cells via chemical or physical processes may enhance the biosorption efficiency for heavy metals. The use of supercritical carbon dioxide (SC-CO 2 ) to inactivate bacteria based on destruction of cell walls took place due to the pressurized CO 2 . In this study, the pre-treatment of Bacillus subtilis cells by SC-CO 2 (BM1), steam autoclaving (BM2), and untreated living cells (BM3) were investigated to show improvement in heavy metal removal. The removal process was optimized based on the main factors affecting bacterial biosorption, which included heavy metals ion concentrations, cell biomass concentrations, pH, time, and temperature. The efficiency of bacterial biomass in removing Ni 2þ ions in the presence of different concentrations of Pb 2þ , Cu 2þ , Zn 2þ , and Cd 2þ was also tested. BM1 and BM2 exhibited the highest potential for the removal of nickel ions in comparison to BM3. The maximum efficiency was 98.54, 99.2, and 96.3% for BM1, BM2, and BM3, respectively. Moreover, BM1 displayed a higher biosorption capacity for Ni 2þ , Cu 2þ , Zn 2þ , Pb 2þ , and Cd 2þ (>150 mg g À1 ) than BM2 and BM3. Biosorption on bacterial cell biomass of Ni 2þ ions shows lower removal affinity in the presence of other metal ions.In conclusion, the pre-treatment of bacterial cells biomass by SC-CO 2 enhanced the removal process of heavy metal ions compared to untreated living cells.